Methods and compositions for treating microtubule-mediated viral infections and lesions

ABSTRACT

The present invention provides methods and compositions for preventing or treating virus infections in mammals that utilize microtubule dynamics within mammalian cells. The compositions are applied to mammals via parenteral, oral, anal, aural, ocular, nasal, and topical routes of administration until clinical signs are resolved.

PRIORITY CLAIM

[0001] This application is a continuation of patent application Ser. No.09/506,415-filed Feb. 17, 2000, and claims benefit under 35 U.S.C.§119(e) of prior Provisional Patent Application Serial Number60/120,582-filed Feb. 18, 1999.

FIELD OF THE INVENTION

[0002] This invention relates generally to treatment methods andcompositions comprising naturally occurring or synthetic compounds thatinterfere with the normal structure and function of tubulin and thenormal formation of microtubule structures within a host cell. Thetreatment methods are for preventing or treating virus infections in amammal, and the dermal or mucosal lesions or tumors associated withcertain viral infections in a mammal, that are dependent upon themicrotubule-mediated cytoplasmic transport of a viral genome within thehost cell, or otherwise exploits microtubule dynamics within the hostcell.

BACKGROUND OF THE INVENTION

[0003] Microtubules are one of the primary elements of the cytoskeletalstructure of virtually all eukaryotic cells. Microtubules are hollowpolarized cylinders of approximately 25 nm constructed of proteinheterodimers of α-tubulin and β-tubulin and are typically anchored to amicrotubule-organizing center (MTOC) located near the cell nucleus. Theyhave a dynamic fast-growing “plus-end” and a slower growing “minus-end”.The tubulin sub-units are moved toward the minus-end of the microtubulewhen additional tubulin sub-units polymerize at the plus-end, thuscreating a “treadmilling” effect.

[0004] Microtubule associated proteins (MAPs) are involved in themovement of materials across the cytoplasm. Dynein is a minus-enddirected MAP, and kinesin is a plus-end directed MAP. Dynein isassociated with the movement of materials across the cytosol from thecell membrane toward the nuclear pore complex. One domain of the dyneinMAP attaches to the cargo and another domain attaches to a tubulinsub-unit. As the tubulin sub-unit moves toward the minus-end of themicrotubule, it carries the cargo along with it. Kinesin is associatedwith the movement of materials across the cytosol from the nucleustoward the cell membrane.

[0005] Viral genomes are one example of materials that are transportedacross the cytoplasm of a host cell by normally functioning microtubuledynamics. For example, recent studies investigating the mechanisms ofviral transport in the cytoplasm demonstrate that herpesvirusreplication is dependent on the normal functioning of microtubuledynamics within the host cell. An incoming HSV capsid binds to themicrotubule at the plus end as described herein and is transportedtoward the minus-end where it is released and enters the cell nucleus tobegin the replication process.

[0006] These observations suggest a novel approach to the development ofeffective antiviral therapies comprising pharmacological agents thatinterfere with the normal structure or function of microtubules withinmammalian cells. To the best of the applicant's knowledge, suchantiviral therapies have not heretofore been identified or described.

[0007] Researchers have observed that a number of viruses are dependentupon microtubule-mediated cytosolic transport for replication. Examplesof these may include, but not limited to, Herpes Simplex 1 (HSV-1),Herpes Simplex 2 (HSV-2), Cytomegalovirus (CMV), Varicella-Zoster virus(VCV), Epstein Barr virus (EBV), Herpes Simplex 6 (HSV-6), HerpesSimplex 7 (HSV-7), Herpes Simplex 8 (HSV-8), Papilloma virus (PPV),Vaccinia virus (VV), Adenovirus, Parvovirus, Human Immunodeficiencyvirus (HIV), and rabies virus.

[0008] Examples of compounds that have been identified asanti-microtubule agents include, but are not limited to, taxanes andtaxoids, discodermolide, epothilones A and B, eleutherobin,taccalonolide, colchicine, colcemid, demecolcine, the vinca alkaloidsincluding vincrisitine, vinepidine, vindesine, vinblastine, vinorelbine,desformyl vincrisitine, desacetyl desformyl vincristine, and vinflunine,phomopsin A, ustiloxins, cryptophycins, halichondrins, estramustine,rhizoxin, nocodazole, and any analogues or derivatives of any of theabove.

[0009] Taxanes (e.g., paclitaxel, docetaxel), discodermolide,epithilones A and B, eleutherobin, and taccalonolide, are examples of anovel class of anti-microtubule agents that share the ability tostabilize microtubules by inducing tubulin polymerization and inhibitingmicrotubule disassembly.

[0010] Paclitaxel (also known under the trademark Taxol®) is perhaps themost familiar of the taxanes. It was first isolated in 1971 from thebark of Taxus brevifolia, commonly known as the Pacific Yew, and wasapproved in 1992 by the US Food and Drug Administration for treatment ofmetastatic ovarian cancer and later for breast cancer. Its mechanism ofaction is believed to involve promoting formation and hyperstabilizationof microtubules, thereby preventing the disassembly of microtubulesnecessary for completion of cell division. It also has been reportedthat paclitaxel induces expression of cytokines, affects the activity ofkinases and blocks processes essential for metastasis, in as yetuncharacterized mechanisms of action.

[0011] Paclitaxel has attracted unusually strong scientific attention,not only because of its unique antiproliferative mechanism of action,but also because it is active against nearly all cancers against whichit has been tested, and because it has been discovered to be an analogof numerous, closely-related compounds occurring naturally. Taxanes arenow recognized as an important new class of anticancer compounds.

[0012] Eleutherobin was originally discovered in rare soft corralsbelonging to the family eleutherobia collected from a specific region ofthe Indian Ocean near Australia. It has since been synthesized byscientists at The Scripps Research Institute. Eleutherobin has amechanism of action similar to paclitaxel, i.e., the stabilization ofmicrotubules and the inhibition of microtubule disassembly.

[0013] Epithilones were originally isolated from a species of soilbacteria collected from the banks of the Zambezi River in the Republicof South Africa, and researchers at The Scripps Research Institute havesynthesized derivatives of these compounds. Epothilones are ofparticular interest because they are more soluble in water thanpaclitaxel, have higher activity, and are more easily available (i.e.,from a cellulose degrading bacterium). They have been shown to displacepaclitaxel from its binding site in β-tubulin, and have demonstrated amechanism of action similar to that of paclitaxel.

[0014] Discodermolide was originally isolated from the spongeDiscodermia dissoluta and has now been totally synthesized.Discodermolide binds to tubulin dimers in microtubules and induces thepolymerization of tubulin similar to paclitaxel, and is perhaps evenmore potent as anti-microtubule agent.

[0015] Colchicine, colcemid, demecolcine, the vinca alkaloids includingvincrisitine, vinepidine, vindesine, vinblastine, vinorelbine, desformylvincrisitine, desacetyl desformyl vincristine, and vinflunine, phomopsinA, ustiloxins, cryptophycins, halichondrins, estramustine, rhizoxin, andnocodazole are examples of a novel class of anti-microtubule agents thatshare the ability to interfere with normal microtubule dynamics byinhibiting the polymerization of tubulin within a host cell, thuspreventing the formation of microtubules.

[0016] Colchicine is a water-soluble alkaloid found in the autumncrocus. The vinca alkaloids, vinblastine and vincristine, are derivedfrom the Madagascar periwinkle, and vindesine and vinorelbine aresemisynthetic derivatives of vinblastine. These alkaloids alsoapparently interfere with a cell's ability to synthesize DNA and RNA.The ustiloxins and phomopsins are a family of tubulin-binding cyclicpeptides, which have shown potent in vitro anti-tumor activity,particularly against human breast and lung cancer cell lines. Rhizoxinis produced by the fungus Rhizopus chinensis, and has also demonstratedthe ability to inhibit the polymerization of tubulin into microtubules.

[0017] Thus, there is an unmet need to have an anti-viral treatmentrepertoire using a plurality of compositions against a plurality ofviruses that target microtubule related processes.

SUMMARY OF THE INVENTION

[0018] The invention provides the treatment repertoire using effectiveamounts of at least one composition of the plurality of compositionsthat target microtubule related processes that are introduced to mammalsvia parenteral, oral, nasal, anal, aural, ocular, and topical routes ofadministration. The parenteral routes of administration includesintervascular injections, intermuscular injections, interdermalinjections, subdermal injections, interspinal injections, andintercerebral injections. The intervascular injections further includeintravenous and interarterial injections.

[0019] The invention provides formulations comprising an effectiveamount of one or more pharmacological agents known to interfere with thenormal structure or function of microtubules. The invention describes anapplication of these formulations to mammalian cells, either topicallyor systemically, for the purpose of preventing or treating viralinfections and the dermal or mucosal lesions or tumors associated withviral infections.

[0020] The present invention provides compositions and methods fortreating diseased, biological tissue, such as the epidermis, in mammals.The plurality of compositions of the present invention can be used totreat epidermal lesions, such as those resulting from viral infectionsincluding, but not limited to: HSV-1, HSV-2, HSV-6, HSV-7, HSV-8, VZV,CMV, EBV, and PPV. The plurality of compositions of the presentinvention can also be used to treat epidermal lesions, ulcerations,abrasions, inflammation and other conditions resulting from microbialinfections. In particular, compositions of the present invention areespecially adapted to treat lesions caused by herpes viruses.

[0021] The effective amounts of at least one of the compositions of aplurality of compositions applied to a mammal is determined by assessingwhether virus reductions in virus infections are manifested asimprovements in clinical signs presented by the mammal, and re-applyingthe effective amount until reduction in virus infections are manifestedas improvements in clinical signs presented by the mammal. Howcompositions are applied depend upon the intended route ofadministration used in introducing the effective amounts of compositionsthat target microtubule related processes. The effective amounts dependsupon composition stabilities, concentrations, and solubilities, andwhether single or more than one composition is administered viaparenteral, oral, anal, aural, nasal, ocular, and topical routes ofadministration.

[0022] In one embodiment, the present invention provides compositionsuseful for treating diseased, biological tissue, such as the epidermis,in mammals. The compositions of the present invention are effective intreating viral infections and inflammation and lesions associated withviral infections. The compositions of the present invention include atleast one anti-microtubule agent. The anti-microtubule agent can be anaturally occurring compound, a semi-synthetic compound, or can be anentirely synthetic compound that is chemically synthesized by any meansknown to those skilled in the art.

[0023] Examples of compounds that have been identified asanti-microtubule agents include, but are not limited to, taxanes,taxoids, discodermolide, epothilones A and B, eleutherobin,taccalonolide, colchicine, colcemid, demecolcine, the vinca alkaloidsincluding vincrisitine, vinepidine, vindesine, vinblastine, vinorelbine,desformyl vincrisitine, desacetyl desformyl vincristine, and vinflunine,phomopsin A, ustiloxins, cryptophycins, halichondrins, estramustine,rhizoxin, and nocodazole. It is appreciated that the examples ofcompounds include any analogues or derivatives of the foregoinganti-microtubule agents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The preferred and alternative embodiments of the presentinvention are described in detail below with reference to the followingdrawings.

[0025] The foregoing aspects of many of the attendant advantages of thisinvention will become more readily appreciated as the same becomesbetter understood by reference to the following detailed description,when taken in conjunction with the accompanying drawings, wherein:

[0026]FIG. 1A is a Drug Screening Report for TBT which lists theanalytical results for bioactive effectiveness of TBT in terms of CPE,EC₅₀, CC₅₀, IC₅₀, SI=CCSO/EC₅₀; Cytopathic Effect Inhibition and PlaqueReduction Assays For HSV-1, HSV-2, CMV, MCMV, and VSV, in HFF cellcultures are detailed;

[0027]FIG. 1B is a Drug Screening Report for TBT which lists theanalytical results for bioactive effectiveness of TBT in terms of CPE,EC₅₀, CC₅₀, IC₅₀, SI=CC₅₀/EC₅₀; Cytopathic Effect Inhibition and PlaqueReduction Assays For EBV in DAUDI cell cultures are detailed; ToxicityAssays in HFF and DAUDI cell cultures are detailed;

[0028]FIG. 2 is a descriptive listing of the HSV ELISA Method proceduralsteps used in measuring the anti-viral effectiveness of TBT to variousHSV-1 and HSV-2 strains;

[0029]FIG. 3 represents the analytical IC₅₀ results for HSV-1 viralantigens, specifically, for HSV-1 control (ATCC #VR-733, Strain F),which demonstrated a TBT-IC₅₀=3 μg/ml;

[0030]FIG. 4 represents the analytical IC₅₀ results for HSV-1 viralantigens, specifically, for HSV-1, clinical specimen #1 (TBT-IC₅₀=3μg/ml);

[0031]FIG. 5 represents the analytical IC₅₀ results for HSV-1 viralantigens, specifically, for HSV-1, clinical specimen #2 (TBT-IC₅₀=0.25μg/ml);

[0032]FIG. 6 represents the analytical IC₅₀ results for HSV-2 viralantigens, specifically for HSV-2 control (ATTC #VR-734 , Strain G),which demonstrated a TBT-IC₅₀=0.75 μg/ml;

[0033]FIG. 7 represents the analytical IC₅₀ results for HSV-2 viralantigens, specifically for HSV-2, clinical specimen #3 (TBT-IC₅₀=2.0μg/ml);

[0034]FIG. 8 represents the analytical IC₅₀ results for HSV-2 viralantigens, specifically for HSV-2, clinical specimen #4 (TBT-IC50=0.25μg/ml);

[0035]FIG. 9 represents the analytical IC₅₀ results for HSV-2 viralantigens, specifically for clinical specimen #5 ( TBT-IC₅₀=0.5 μg/ml);and

[0036]FIG. 10 represents the analytical IC₅₀ results for HSV-2 viralantigens, specifically for clinical specimen #6 (TBT-IC50<0.10 μg/ml).

DETAILED DESCRIPTION OF THE INVENTION

[0037] The invention pertains to methods and pharmaceutical compositionsfor preventing or treating viral infections in a mammal, and the dermalor mucosal lesions or tumors caused by viral infections in mammalsincluding, but not limited to, human herpesvirus infections (HHV), andmore preferably primary or recurrent HSV infections.

[0038] In one aspect of the invention, primary and recurrent lesions,sores, or tumors of the skin and mucosa are treated with a topicalcomposition comprising an effective amount of an anti-microtubule agentto a human suffering from herpesvirus infections. The area to be treatedmay include, the lips, eyes, mouth, genital and anal area, and otherareas accessible to topical administration, which may be the site of aherpes lesion, sore, or tumor.

[0039] In a second aspect of the invention relates to a method oftreating or preventing herpesvirus infections using pharmaceuticalcompositions that contain an effective dosage of an anti-microtubuleagent which is administered to a patient suffering from viralinfections, or at risk for contracting viral infections, in order totreat or prevent the viral infection in the patient.

[0040] In a third aspect of the invention, primary and recurrentinfections caused by a herpesvirus that may or may not be associatedwith lesions, are treated with a pharmaceutical composition or acombination of pharmaceutical compositions that is used for regional orsystemic administration of an inhibitory effective amount of ananti-microtubule agent contained in the pharmaceutical composition oranti-microtubule agents contained in the combination of pharmaceuticalcompositions. These types of infections would include viral causedneonatal diseases, encephalitis diseases, and respiratory distresssyndrome or acuteonset bronchospasm diseases.

[0041] The pharmaceutical compositions of the invention contain anantiviral agent. For the purpose of this invention, the antiviral agentis any molecule from the pharmaceutical composition that interferes withthe normal structure and function of microtubule dynamics within a hostcell. The antiviral agent interferes with the viability, production, oractivity of tubulin proteins within a host cell, including the promotionor inhibition of microtubule polymerization, or the promotion orinhibition of microtubule disassembly. Additionally, the antiviral agentmay also exhibit inhibitory effects upon MAPs that mediate the transportof viral particles including a viral genome within the host cell.

[0042] The scope of the invention includes any pharmaceuticalcomposition that modifies the activity or effect of microtubule-mediatedcytoplasmic transport of viral particles, either directly or indirectly,in decreasing the permissiveness of cells to virus infection. Thespecific embodiments of this invention as described herein are notintended to limit the applicability of the principles involved. Thoseskilled in the art are aware that there are, or may be, other means ofmodifying the activity of microtubules.

[0043] The present invention provides pharmaceutical compositions whichmay contain between 0.005% and 30%, (weight percentage) of the antiviralagent as described above and of one or more of a plurality ofpharmaceutically acceptable excipients. Among the plurality ofpharmaceutically acceptable excipients include lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, mircrocrystallinecellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup andmethyl cellulose.

[0044] The pharmaceutical compositions can additionally include:lubricating agents such as talc, magnesium stearate and mineral oil;wetting agents; emulsifying and suspending agents; preserving agentssuch as methyl-benzoates and proplyhydroxy-benzoates; sweetening agentsand flavoring agents.

[0045] The invention can be formulated so as to provide quick, sustainedor delayed release of the antiviral agent after administration to thepatient by employing procedures known in the art. Other components maybe added to the pharmaceutical composition based upon the related drugdelivery system, to improve the pharmacokinetics or pharmacodynamics ofthe composition.

[0046] In making the compositions of the invention, the antiviral agentis usually mixed with and/or diluted by, one or more excipients.Alternatively, the antiviral agent may be enclosed within a carrier, inthe form of a capsule or other container. As the excipient may be solid,semi-solid, or liquid, the resultant composition may also be solid,semi-solid, or liquid.

[0047] The pharmaceutical compositions of the present invention caninclude other active ingredients. For instance, drugs that are commonlyused to treat herpes, such as nucleoside analogs, or pain relievingdrugs such as acetaminophen may be added to the composition.Additionally, agents or chemical additives that enhance the antiviralactivity of the agent, or more than one antiviral agent, can be includedin the pharmaceutical composition. It may also be desirable to includeone or more penetrating agents, such as dimethyl sulfoxide (DMSO), toconvey the active ingredients into the epidermal tissues.

[0048] Topical pharmaceutical compositions in the form of an ointment,cream, gel, solution, lotion, emulsion, aerosol, powder, or othertopical vehicle, including a sponge, suppository or stick, are designedand prepared such that a therapeutically effective amount of theantiviral agent is brought into contact with diseased tissue. Anexcipient or carrier may take a wide variety of forms depending on theform of preparation desired for topical administration, which includesrectal or vaginal administration. In preparing pharmaceuticalcompositions in topical dosage form, any of the usual pharmaceuticalmedia may be used.

[0049] To create a viscous ointment, for example, de-ionized water, oiland an emulsifier are intermingled to create an emulsion. An oilsuitable for such a purpose is petrolatum registered by the UnitedStates Pharmacopia/National Formulary (USP/NF). A wax suitable as anemulsifier is wax registered by the USP/NF. A preservative ointmentincludes USP/NF registered methyl paraben or propyl paraben andhumectants, such as propylene glycol.

[0050] Solid compositions such as tablets, pills and capsules may beprepared by mixing the antiviral agent with an excipient so that theantiviral agent is evenly distributed throughout the composition, thusmaking it possible to subdivide the composition into equally effectivedoses.

[0051] The antiviral compositions of the present invention may beincorporated into liquid forms to be administered orally or by injectionincluding aqueous or non-aqueous solutions, syrups, aqueous or oilsuspensions and emulsions comprising edible oils, such as corn oil, aswell as tinctures and similar pharmaceutical vehicles.

[0052] Compositions for inhalation may include solutions and suspensionsin pharmaceutically acceptable, aqueous or organic solvents or powders.The liquid or solid compositions may contain pharmaceutically acceptableexcipients as described herein. Administration of the compositionsthrough the oral or nasal respiratory tract is preferred for local orsystemic effect.

[0053] Although certain preferred embodiments of the invention have beendescribed herein, these are not meant to limit the invention, whichcovers all alternatives, modifications and equivalents as may beincluded within the scope of this invention. The preferred embodimentspresented herein are represent sent the most useful embodiments of theinvention, as well as a description of the underlying principles andconceptual aspects of the invention.

[0054] Treatment with the pharmaceutical compositions of this inventionmay begin when a patient has been exposed to a virus, when symptoms ofinfection, such as epidermal lesions, are apparent in a patient, or whena patient is diagnosed with an active viral infection. Treatment shouldbe continued until risk of infection is over or until the activesymptoms of the viral infection have subsided.

[0055] As will be apparent to those skilled in the art, variousmodifications, adaptations and variations of the preceding and foregoingspecific disclosure can be made without departing from the scope of theinvention claimed herein. The following examples are intended only toillustrate and describe the invention rather than limit the claims whichfollow.

[0056] The herpes simplex viruses, including HSV-1 (oro-facial) andHSV-2 (genital herpes), present a serious problem for millions of peopleworldwide. To the best of applicant's knowledge, until now, aneffective, economical, and readily available topical treatment has notexisted.

[0057] Herpes simplex virus is a common, recurrent, and chronicinfection. It is estimated that at least 75% of the world's populationhas been infected with HSV-1 and more than 20% with HSV-2. Although themajority of cases are asymptomatic, chronic outbreaks of lesions arevery common, usually occurring in mucous membrane areas and thesurrounding skin. The most common of these lesions occur on the lips orface and are commonly referred to as “cold sores” or “fever blisters.”Genital herpes lesions occur on the genitals and buttocks and areparticularly troubling because of their possible role in contributing tothe spread of HIV.

[0058] Herpes lesions first appear as an area of irritation (an itchingor burning sensation) known as the prodromal stage. Within a few hours,these lesions develop into small vesicles or blisters. Typically, thesevesicles soon rupture and form shallow ulcerations which may scab overand heal in about ten to twenty days. The ruptured vesicles may alsocause secondary infections and spread the virus to the surroundingtissue.

[0059] After initial exposure to the herpes simplex virus, the hostdevelops antibodies that can maintain the virus in a latent state.Despite the presence of antibodies, the latent virus may be reactivatedby stress, exposure to sunlight, fever, hormonal changes, menstruation,and trauma. Eruptions can occur randomly and may persist for weeks.

[0060] Research supported in part by the National Institute of Allergyand Infectious Diseases (and conducted by virologists at the Universityof Chicago and the University of Alabama, has demonstrated thatcompounds comprising at least one antimicrotubule agent possessclinically significant anti-viral properties that specifically inhibitreplication of HSV-1 and HSV-2 in vitro.

[0061] There is currently no known cure for herpes simplex virusinfections. However, a topical therapy that deliversclinically-demonstrated, anti-viral compositions to the affected area,inhibiting viral replications in the lesions, accelerating healing ofthe existing lesion, and preventing the spread of secondary infections,would be of enormous benefit to the herpes sufferer.

[0062] In one embodiment, the present invention provides compositionsuseful for treating diseased, biological tissue, such as the epidermisor mucous membranes, in a mammal. The methods and compositions of thepresent invention are effective in treating viral infections, dermal ormucosal lesions, inflammation, or tumors associated with said viralinfections. The compositions of the present invention include at leastone anti-microtubule agent. The anti-microtubule agent can be anaturally occurring compound, a semi-synthetic compound, or can be anentirely synthetic compound that is chemically synthesized by any meansknown to those skilled in the art.

[0063] Examples of compounds that have been identified asanti-microtubule agents include, but are not limited to, taxanes andtaxoids, discodermolide, epothilones A and B, eleutherobin,taccalonolide, colchicine, colcemid, demecolcine, the vinca alkaloidsincluding vincrisitine, vinepidine, vindesine, vinblastine, vinorelbine,desformyl vincrisitine, desacetyl desformyl vincristine, and vinflunine,phomopsin A, ustiloxins, cryptophycins, halichondrins, estramustine,rhizoxin, nocodazole, and any analogues or derivatives of any of theabove.

[0064] A presently preferred composition of the present invention isformed from an extract from the Pacific Yew tree (Taxus brevifolia)combined with virgin olive oil and beeswax. The T. brevifolia extract isextracted by the method set forth in Example 1 herein and combined witholive oil at a ratio of about 1:1. The ethanol and water (from theextract) are completely evaporated before combining with the beeswax ata ratio of about 6:1. High Performance Liquid Chromatography (H.P.L.C.)analysis of the foregoing T. brevifolia extract revealed the presence of8.1 μg/ml paclitaxel, 77.87 μg/ml cephalomannine, and 623.79 μg/ml10-deacetyl-7-xylosyltaxol, plus some other taxanes present in minoramounts. Preferably, the compositions of the present invention aretopically applied in the form of an ointment, salve or lotion to thesite of disease. Compositions of the present invention can be mixed withother physiologically acceptable components, such as carriers,stabilizers or antioxidants, to form an ointment, salve or lotion havingdesirable physical and chemical properties, and consistency. See,Remington's Pharmaceutical Sciences, 16th Edition, Osol, A., Ed (1980).

[0065] Preferably, compositions of the present invention include atleast one anti-microtubule agent in an amount of from about 0.005% toabout 30% of the total weight of the composition. Preferably, thecompositions of the present invention also include a natural oil suchas, but not limited to, olive oil, mineral oil, corn oil, sunflower oil,peanut oil, and fish oil. Preferably, the compositions of the presentinvention also contain a wax such as, but not limited to, beeswax,U.S.P. Carbowax 5000®, U.S.P. Carbowax 6000® (the foregoing Carbowax®products are manufactured by Union Carbide Corporation, WorldHeadquarters, 39 Old Ridgebury Road, Danbury, and Conn. 06817-001) andpetrolatum.

[0066] In addition to the foregoing components, compositions of thepresent invention can include additional ingredients including, but notlimited to, analgesics and anesthetics.

[0067] The compositions of the present invention, when appliedtopically, soothe the discomfort associated with viral lesions and otherepidermal conditions, prevent the lesion from cracking or bleeding,reduce the time to healing, and prevent the spreading of viralinfections by effectively inhibiting viral replication.

[0068] In another embodiment, the present invention includes a method oftreating diseased biological tissue, such as the epidermis or mucousmembranes, in a mammal. The method of the present invention includes thestep of contacting a diseased biological tissue, such as the epidermis,with a composition of the present invention containing an amount of anantimicrotubule agent, or related compound, that is effective toameliorate the disease symptoms. The methods and compositions of thepresent invention are effective in treating viral infections, dermal ormucosal lesions, inflammation, or tumors associated with said viralinfections. Examples of viral infections that can be treated using thecompositions and method of the present invention include, but notlimited to, HSV-1, HSV-2, HSV-6, HSV-7, HSV-8, VZV, CMV, EBV, and PPV.

[0069] The compositions of the present invention should be applieddirectly to the affected portion of the mammalian body, such as theepidermis. Preferably, in the practice of the method of the presentinvention, the quantity of a composition of the present invention thatis applied to an affected bodily surface is sufficient to cover theaffected area. A sufficient quantity of a composition of the presentinvention should preferably be reapplied as often as is necessary tokeep the affected area covered until the condition has completelycleared. In the case of viral infections such HSV-1 or HSV-2, acomposition of the present invention should be applied at the very first(prodromal) indication of symptoms (i.e., burning, itching, or tinglingsensations). Such early application will, in many cases, prevent lesionsfrom fully developing or spreading, thus significantly limiting the timeto healing, discomfort, risk of further infection to self and others,and risk of infection from other opportunistic viruses such as HIV.

[0070] The following examples merely illustrate the best mode nowcontemplated for practicing the invention, but should not be construedto limit the invention. Examples 1 and 2 describe methods of manufactureof various forms of the preferred embodiment. Examples 4-6 describe theclinical effectiveness of preferred embodiments applied to patientsinfected with HSV-1 and HSV-2. Examples 7 and 8 demonstrate the specificanti-viral activity that Taxus brevifolia Tinctures (TBT) exhibits invarious viral infected cell culture systems.

EXAMPLE 1

[0071] Extraction of Naturally-Occurring Taxanes from Yew

[0072] Needles and branches of yew tree species are harvested by pruningthe terminal branch tips of the selected species in such a way as toencourage new growth and preserve the tree for future harvesting, thusmaintaining the existing biomass as a fully renewable resource. Thematerial is then milled in order to increase the amount of exposedsurfaces and render the material more compact. One part (by weight) ofthe material is placed in a suitable container and saturated with twoparts (by weight) of ethanol (or other suitable solvent such asisopropyl alcohol, butanol, or methanol in concentrations ranging from5% to 100%). The resulting mixture is allowed to macerate in the solventfor a specific time (typically 7 to 14 days) until the material isexhausted of its constituents, and then is hydraulically pressed andfiltered to remove the residue of plant material.

EXAMPLE 2

[0073] Second Exemplary Method for Extraction of Naturally-OccurringTaxanes from Yew

[0074] The raw materials are harvested as described in Example 1 andplaced in a columnar percolator. The material may be pre-moistened forseveral hours in a fraction of the solvent and then passed through acoarse sieve and lightly packed in the chamber, with a wad of gauzebelow and filter paper above. The drain is closed and sufficient solventis added to cover the material. The vessel is then covered and allowedto macerate for approximately 24 hours. The drain is then opened andfluid is allowed through at the rate of 10 to 30 drops per minute,solvent being added to the top as needed until the material isexhausted. The material is then hydraulically pressed to extract anyremaining fluid which is then added to the percolate.

EXAMPLE 3

[0075] Effectiveness of the Compositions of the Present Invention inTreating Cold Sores

[0076] An adult female, suffering from severe, recurrent HSV-1infections, applied the presently preferred composition of the inventionto a labial infection (cold sore) after it had developed into a largeblister (the presently preferred composition of the invention is a salveprepared from a T. brevifolia extract, prepared by the method set forthin Example 1 herein, which is combined with olive oil at a ratio ofabout 1:1. The ethanol and water (from the extract) are completelyevaporated before combining T. brevifolia extract and olive oil withbeeswax at a ratio of about 6:1.). The composition was reappliedregularly as needed to keep the blister covered. The blister disappearedwithin 24 hours and was replaced by healthy tissue. Several monthslater, the subject experienced prodromal symptoms (i.e., tingling anditching) and applied the compound immediately. Again, the compositionwas reapplied regularly for 24 hours. The infection did not progressfurther and produced no lesion or other evidence of infection.

EXAMPLE 4

[0077] Effectiveness of the Compositions of the Present Invention inTreating Genital Herpes in a Female Subject

[0078] An adult female, suffering from mild, recurrent HSV-2 infectionsthat typically produced lesions on the genitalia lasting approximately 7days, applied the presently preferred composition of the presentinvention to the affected parts immediately upon experiencing prodromalsymptoms. The infection did not progress further and produced no lesionsor other evidence of infection.

EXAMPLE 5

[0079] Effectiveness of the Compositions of the Present Invention inTreating Genital Herpes in a Male Subject

[0080] An adult male, suffering from moderately severe, recurrent HSV-2infections that typically produced lesions on the genitalia lastingapproximately 7 to 10 days, applied the presently preferred compositionof the invention to the affected parts immediately upon experiencingprodromal symptoms. The composition was reapplied regularly as needed tokeep the affected area covered. The prodromal symptoms were resolvedwithin 48 hours. The infection did not progress further and produced nolesions or other evidence of infection.

EXAMPLE 6

[0081] Effectiveness of the Compositions of the Present Invention inTreating Genital Herpes in a Male Subject

[0082] An adult male, suffering from moderately severe, recurrent HSV-2infections that typically produced lesions on the genitalia lastingapproximately 8 days, applied the presently preferred composition of theinvention to the affected parts immediately upon experiencing prodromalsymptoms. Some lesions did appear and the composition was reappliedregularly as needed to keep the affected area covered. All symptoms wereresolved within 4 days.

EXAMPLE 7

[0083] Screening Assays for Activity of TBT (ARB ID# 980332) AgainstHSV-1, HSV-2, CMV, VZV, and EBV

[0084] The data below, discloses the results of ELISA assaysdemonstrating the effectiveness of the Taxus Brevifolia Tinctures (TBT)extracts (Lot ARB-ID# 99-332) of Example 1 against Herpes SimplexVirus-1 (HSV-1).

[0085] General Approach for Determining Antiviral Activity and Toxicity

[0086] A. Screening Assays for Activity Against HSV-1, HSV-2, CMV, andVZV

[0087] All the screening assay systems utilized have been selected toshow specific inhibition of a biologic function, i.e., cytopathic effect(CPE) in susceptible human cells. In the CPE, inhibition assay, drug isadded 1 hr prior to infection so the assay system will have maximumsensitivity and detect inhibitors of early replicative steps such asabsorption or penetration as well as later events. To rule outnon-specific inhibition of virus binding to cells all compounds thatshow reasonable activity in the CPE assay are conformed using aclassical plaque reduction assay in which the drug is added 1 hr afterinfection. In the case where a compound blocks attachment, it will showup positive in the CPE assay, but may be negative by plaque assay. Inthis case, the plaque assay is repeated with drug being added prior toviral infection. Using this approach, we have been able to identifycompounds that inhibit virus absorption. These assay systems also can bemanipulated by increasing the pretreatment time in order to demonstrateantiviral activity with oligodeoxynucleotides and/or peptides and bydelaying addition of drug after infection, information regarding whichstep in the virus life cycle is inhibited (i.e., early vs. latefunctions) can be gained.

[0088] 1. Efficacy. In all the assays used for primary screening, aminimum of six drug concentrations were used covering a range of 100μg/ml to 0.03 μg/ml, in 5-fold increments. From these data, we calculatethe dose that inhibits viral replication by 50% (effective concentration50; EC₅₀) using the computer software program MacSynergy II by M. N.Prichard, K. R. Asaltine, and C. Shipman, Jr., University of Michigan,Ann Arbor, Mich.

[0089] 2. Toxicity. The same drug concentrations used to determineefficacy are also used on uninfected cells in each assay to determinetoxicity of each experimental compound. The drug concentration that iscytotoxic to cells as determined by their failure to take up a vitalstrain, neutral red, (cytotoxic concentration 50; CC₅₀) was determinedas described above. It is very important to determine the toxicity ofnew compounds on dividing cells at a very early stage of testing. Wehave found that a cell proliferation assay using human foreskinfibroblasts (HFF) cells is a very sensitive assay for detecting drugtoxicity to dividing cells and the drug concentration that inhibits cellgrowth by 50% (IC₅₀) is calculated as described above. In comparisonwith four human diploid cell lines and vero cells, HFF cells are themost sensitive and predictive of toxicity for bone marrow cells.

[0090] 3. Assessment of Drug Activity. To determine if each compound hassufficient antiviral activity that exceeds its level of toxicity, aselectively index (SI) is calculated according to CC₅₀/EC₅₀. This index,also referred to as a therapeutic index, was used to determine if acompound warrants further study. For these studies, a compound that hadan SI of 10 or greater was evaluated in additional assay systems.

[0091] B. Confirmation of Antiviral Activity and Toxicity for HSV, CMVand VZV

[0092] 1. HSV-1 and HSV-2. Compounds that showed activity in theCPE-inhibition assay was confirmed using the plaque reduction assay asdescribed in an earlier section. Susceptibility of additional virusstrains including both lab passaged and clinical isolates was determinedfor selected compounds. A battery of ACV resistant HSV strains were alsoutilized.

[0093] 2. CMV. Compounds that have activity in the CPE-inhibition assaywere confirmed using the plaque reduction assay in HFF cells. A varietyof laboratory, clinical, and GCV resistant isolates are also availablefor testing.

[0094] 3. VZV. Compounds were tested for activity in a plaque reductionassay. A battery of laboratory, clinical, and ACV-resistant isolates areavailable.

[0095] 4. Toxicity. In addition to the toxicity component incorporatedinto each assay system, a standardized cell cytotoxicity assay using avital strain uptake (Neutral Red) was performed using 7 days of drugexposure to confluent non-dividing cells. This assay measures directcell killing (CC₅₀). Inhibition of cell growth (IC₅₀) can also bedetermined by treatment of proliferating cells and then assessing theamount of dye uptake.

[0096] C. Assay Systems for Determining Antiviral Activity Against EBVand Toxicity to Lymphoblastic Cells

[0097] 1. Superinfection of susceptible Burkitt's Lymphoma (BL) cellswith P3HR-1 virus followed by analysis of specific EBV gene productexpression using monoclonal antibodies provides a convenient andrepeatable system of evaluate inhibition of EBV gene expression duringearly and late stages of the virus replication cycle. We can evaluatediffuse (D) and restricted (R) early antigens (EA) as well as viralcapsid antigen (VCA) by fluorescence microscopy and by fluorescence flowcytometry.

[0098] 2. Screening Assay for EBV Activity. The initial system to beused to determine antiviral activity against EBV will be VCA productionDaudie cells using an immunofluorescence assay (IFA). As in all theother assays, six concentrations of drug covering a range of 100 μg/mlto 0.03 μg/ml will be utilized. Using the results obtained fromuntreated and drug treated cells an EC₅₀ can be calculated. Selectedcompounds that have good activity against EBV VCA production withouttoxicity will be tested for their ability to inhibit EBV DNA synthesis.

[0099] 3. Toxicity. In each assay system utilized, drug treatment ofuninfected cells is incorporated to obtain as much toxicity data aspossible.

[0100] 4. Confirmation of drug activity against EBV DNA production usingin situ DNA hybridization assay. All compounds that have an SI>10 in thescreening assay or ones selected by the project offer will be confirmedin a hybridization assay that measures the amount of EBV DNA produced byP3HR-1 infected cells. As in all other assay systems utilized, a widerange of drug concentrations will be utilized so an accurate EC₅₀ can becalculated. Uninfected control cells treated with drug will also beutilized as another measure of drug toxicity.

[0101] a. Infection and drug treatment: 106 cells/tube are infected withEBV at a dilution of 1:40. After incubation for 45 minutes at 37° C., 3ml of RPMI, a cell culture media, is added and the cells pelleted bycentrifugation. The supernatant was then discarded and the cellsresuspended in 4 ml of RPMI needing containing various concentrations ofdrug. After incubation for 48 hours, the cells are counted in each tube,washed with PBS and spotted on slides. The slides are left to air-dryovernight and then fixed in acetone for 10 minutes at room temperature.

[0102] b. DNA hybridization: The biotin labeled EBV probe is added toeach spot and the slide is covered with a glass overslip. The slide isthen heated on a hot plate at 95° C. for three minutes. After heating,the slide is left to sit at room temperature for 20 minutes, for the DNAto anneal. The overslips are then removed and the Post HybridizationReagent is added to each spot. After incubation for 10 minutes andrinsing with washing buffer, Detection Reagent is applied. This is lefton for 20 minutes at room temperature and then washed off with washingbuffer. Chromagen Substrate Solution is added and incubated for 10minutes at room temperature. Washing buffer is used to rinse it off, andthe slides are counter stained for 30-60 seconds with fast Green stain.The slides are then rinsed with deionized water and mounted with water.

[0103] C. Reading and calculation of results: The slides are viewed in alight microscope under a magnification of 100-400. Positive cells appearas pink or red spots. All the cells are counted in several fields. Thefraction of red spots in the total number of cells counted multiplied by100 reflects the percent of hybridization.

EXAMPLE 8

[0104] ELISA Testing Of HSV Susceptibility To TBT

[0105] The data below discloses the results of ELISA assaysdemonstrating the effectiveness of the T. brevifolia tincture extractsof Example 1 against Herpes Simplex Virus (HSV). Susceptibility Testingby ELISA (Enzyme Linked Immunosorbent Assay)

[0106] HFF cells were inoculated into 96-well microtiter trays at adensity of 7×10³ cells per well. The plates were incubated at 37° C. in5% CO₂ until the cells were confluent, usually three days. Sixty wellsof each plate were used: six uninfected cell control wells, sixvirus-infected control wells without drug, and six replicates of eightdilutions of the drug. Dilutions of each virus were prepared in minimalessential media (MEM). The growth medium was removed from all wells and50 μl of MEM was added to the cell control wells and 50 μl of virusinoculum with a multiplicity-of-infection (MOI)˜0.05, was added to theremaining wells. The virus was allowed to absorb for one hour at 37° C.The inoculum was removed and 100 μl of MEM was added to the cell controlwells and the virus control wells. Eight dilutions of Taxus brevifoliaTincture (TBT) were prepared in MEM and 100 μl of the diluted drug wasadded to the remaining wells beginning at a 4.0 μg/ml through a 0.1μg/ml of the drug. All plates were incubated at 37° C. in 5% CO₂.

[0107] After incubating for 48 hours, the plates were examined using aninverted phase contrast microscope to insure that viral CPE was presentin the virus control wells and to score the CPE in all wells of theplate including the virus control wells and all drug dilution wells.Each row of wells was scored from 0 to 4+ and 4+ indicated that allcells showed CPE. This was done to insure that the inhibition of CPEcorrelated with the quantitative ELISA results. The medium was thenremoved from all microtiter wells and 100 μl of a blocking solutionconsisting of 0.5% bovine serum albumin (BSA) in phosphate bufferedsaline (PBS), pH 7.2, was added to each well for 30 min. at roomtemperature. The blocking solution was removed, the cells were fixed byadding 100 μl of ethanol/acetone (95:5, v/v) to each well and the plateswere placed at −20° C. for 30 min. Each well was washed four times with200 μl of wash solution (PBS containing 0.5% BSA and 0.05% Tween 20).

[0108] The antibodies used in ELISA were obtained from Dako Corporation,Carpinteria, Calif., and were prepared by immunizing rabbits with anantigen prepared by sonication and extraction of HSV-1 or HSV-2 infectedrabbit cornea cells. All the virion proteins were present in the antigenpreparation used to produce the antibody. To determine the inhibitoryconcentration (IC₅₀), the rabbit polyclonal antibody to HSV-1 or HSV-2conjugated to horseradish peroxidase was diluted in PBS containing 10%normal rabbit serum. A volume of 100 μl of the antibody was added toeach well and the plates were incubated at 37° C. for two hours. Theantibody was removed and the wells were washed four times as before. Theenzyme substrate, 3, 3′, 5, 5′—tetramethylbenzidine (TMB, Sigrna, ST.Louis, Mo.) was added to each well and the plates were incubated at roomtemperature for 3-4 minutes. The O.D. was determined for the uninfectedcell control wells, the virus control wells, and each drug dilution. Thepercent change in O.D. was calculated as follows: (average drug sampleO.D.—average cell control O.D.)/(average virus control O.D.—average cellcontrol O.D.)×100. The IC₅₀ is defined as the dilution of antiviralcompound that produces a 50% or greater reduction in the O.D. of thecolored substrate product.

[0109] While the preferred embodiment of the invention has beenillustrated and described, as noted above, many changes can be madewithout departing from the spirit and scope of the invention.Accordingly, the scope of the invention is not limited by the disclosureof the preferred embodiment.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method to treat viralinfection in mammals, the method comprising: receiving an effectiveamount of at least one of a plurality of compositions targeting aplurality of microtubule processes in mammalian cells, the effectiveamount being delivered by parental routes of administration to reducenon-dermal viral infections for viruses utilizing the plurality ofmicrotubule processes in mammalian cells.
 2. The method of claim 1,wherein the plurality of compositions targeting the microtubule processin mammalian cells includes taxanes, taxoids, discodermolide,epothilones A, epothilone B, eleutherobin, taccalonolide, colchicine,colcemid, demecolcine, vincrisitine, vinepidine, vindesine, vinblastine,vinorelbine, desformyl vincrisitine, desacetyl desformyl vincristine,vinflunine, phomopsin A, ustiloxins, cryptophycins, halichondrins,estramustine, rhizoxin, and nocodazole.
 3. The method of claim 2,wherein the plurality of compositions further includes solubilizers,parenteral solutions, and analgesics.
 4. The method of claim 3, whereinthe parenteral solutions are water-based and includes saline, lactose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, and polyvinylpyrrodlidone.
 5. Themethod of claim 1, wherein the parental route of administration includesintervascular injections, intermuscular injections, interdermalinjections, interspinal injections, and intercerebral injections.
 6. Themethod of claim 1, wherein the viruses utilizing the microtubule processin mammalian cells includes Herpesvirus-1 (HSV-1), Herpesvirus-2(HSV-2), Cytomegalovirus (CMV), Varacella-Zoster Virus (VZV), EpsteinBarr virus (EBV), Herpes Simplex 6 (HSV-6), Herpes Simplex 7 (HSV-7),Herpes Simplex 8 (HSV-8), human Papilloma Virus (HPV), Vaccinia Virus(VV), Adenovirus, Parvovirus, Human Infectivity Virus (HIV), and rabiesvirus.
 7. The method of claim 1, wherein the parental route ofadministration includes filling a syringe with an effective amount of atleast one of the plurality of compositions to reduce viral infectionsinto the syringe, injecting the effective amount into a mammal,assessing whether virus reductions in virus infections are manifested asimprovements in clinical signs presented by the mammal, and re-injectingthe effective amount until reductions in virus infections are manifestedas improvements in clinical signs presented by the mammal.
 8. A methodto treat viral infection in mammals, the method comprising: receiving aneffective amount of at least one of a plurality of compositionstargeting a plurality of microtubule processes in mammalian cells, theeffective amount being delivered by oral, anal, aural, ocular and nasalroutes of administration to reduce viral infections for virusesutilizing the plurality of microtubule processes in mammalian cells. 9.The method of claim 8, wherein the plurality of compositions targetingthe microtubule process in mammalian cells includes taxanes, taxoids,discodermolide, epothilones A, epothilone B, eleutherobin,taccalonolide, colchicine, colcemid, demecolcine, vincrisitine,vinepidine, vindesine, vinblastine, vinorelbine, desformyl vincrisitine,desacetyl desformyl vincristine, vinflunine, phomopsin A, ustiloxins,cryptophycins, halichondrins, estramustine, rhizoxin, and nocodazole.10. The method of claim 8, wherein the plurality of compositions furtherincludes solubilizers, solutions, and analgesics.
 11. The method ofclaim 10, wherein the solutions are water-based and includes saline,lactose, sucrose, sorbitol, mannitol, starches, gum acacia, calciumphosphate, alginates, tragacanth, gelatin, calcium silicate, cellulose,methyl cellulose, microcrystalline cellulose,polyvinylpyrrodlidoneincludes and syrup.
 12. The method of claim 8,wherein the viruses utilizing the microtubule process in mammalian cellsincludes Herpesvirus-1 (HSV-1), Herpesvirus-2 (HSV-2), Cytomegalovirus(CMV), Varacella-Zoster Virus (VZV), Epstein Barr virus (EBV), HerpesSimplex 6 (HSV-6), Herpes Simplex 7 (HSV-7), Herpes Simplex 8 (HSV-8),human Papilloma Virus (HPV), Vaccinia Virus (VV), Adenovirus,Parvovirus, Human Infectivity Virus (HIV), and rabies virus.
 13. Themethod of claim 8, wherein the oral, anal, aural, ocular, and nasalroutes of administration includes filling a container with an effectiveamount of at least one of the plurality of compositions for reducingviral infections, transferring the effective amount from the containerinto a mouth, anus, ear, eye or nose of a mammal, assessing whethervirus reductions in virus infections are manifested as improvements inclinical signs presented by the mammal, and re-transferring theeffective amount into the mouth, anus, ear, eye or nose of the mammaluntil reductions in virus infections are manifested as improvements inclinical signs presented by the mammal.
 14. A method to treat viralinfection in mammals, the method comprising: receiving an effectiveamount of at least one of a plurality of compositions targeting amicrotubule process in mammalian cells, the effective amount beingdelivered by a topical route of administration to reduce viralinfections in dermal lesions and inflamed areas for viruses utilizingthe microtubule processes in mammalian cells.
 15. The method of claim14, wherein the plurality of compositions targeting the microtubuleprocess in mammalian cells includes taxanes, taxoids, discodermolide,epothilones A, epothilone B, eleutherobin, taccalonolide, colchicine,colcemid, demecolcine, vincrisitine, vinepidine, vindesine, vinblastine,vinorelbine, desformyl vincrisitine, desacetyl desformyl vincristine,vinflunine, phomopsin A, ustiloxins, cryptophycins, halichondrins,estramustine, rhizoxin, and nocodazole.
 16. The method of claim 14,wherein the plurality of compositions further includes solubilizers,lubricants, emulsifiers, waxes, solutions, preservatives, humectants,and analgesics.
 17. The method of claim 16, wherein the solutionsincludes parenteral are water-based and includes saline, lactose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrodlidonenormal, andsyrup.
 18. The method of claim 16, wherein the solubilizers includesdimethy sulfoxide (DMSO), alcohol, petrolatum, and corn oil.
 19. Themethod of claim 14, wherein the viruses utilizing the microtubuleprocess in mammalian cells includes Herpesvirus-1 (HSV-1), Herpesvirus-2(HSV-2), Cytomegalovirus (CMV), Varacella-Zoster Virus (VZV), EpsteinBarr virus (EBV), Herpes Simplex 6 (HSV-6), Herpes Simplex 7 (HSV-7),Herpes Simplex 8 (HSV-8), human Papilloma Virus (HPV), Vaccinia Virus(VV), Adenovirus, Parvovirus, Human Infectivity Virus (HIV), and rabiesvirus.
 20. The method of 14, whereby the topical route of administrationcomprises contacting the dermal lesions and inflamed areas with at leastone of the plurality of compositions, rubbing the composition into thedermal lesions and inflamed areas, and recontacting and re-rubbing thedermal lesions and inflamed areas until the dermal lesions and inflamedareas are resolved.